1. Center of Excellence in Cancer Research Principles of flow cytometry
Tanta University
Principles of flow cytometry
(SAAI)

2. Supervisors: Prof. Mohamed Labib Salem, PhD
Prof. of Immunology; Executive Director, Center of Excellence in Cancer Research, Tanta University
2. Prof. Mohamed Attia Saad, MD
Prof. of Clinical Pathology; Director of Flow Cytometry Unit, Center of Excellence in Cancer Research, Tanta University.

3. Instructor : Mona F. Zidan, BCs
Master Researcher; Research Assistant, Flow cytometry Unit, Center of Excellence in Cancer Research, Tanta University
Approved by the ICGEB to attened the international theoretical and practical training course "Flow cytometry and cell sorting in Biotechnology and Biomedicine Research" which was held at institut Pasteur de Montevideo, Montevideo, Uruguay, Latin America

5. 3. Acquisition process 4. Analysis of data SAAI: Main objectives
By the end of this session you will learn:
Basics of flow cytometry
2. Staining process
3. Acquisition process
4. Analysis of data
5. Interpretation of data

6. Acquisition process Analysis of data SAAI: Main objectives
By the end of this session you will learn:
Basics of flow cytometry
Staining process
Acquisition process
Analysis of data
Interpretation of data

7. Flow Cytometry: Definition
Flow ~ in motion
Cyto ~ cell
Metry ~ measure
Measuring properties of single cells in a fluid stream
Gives us the ability to analyze many properties of many cells in very little time

8. Three main basis: Flow Cytometry: Basis Single cell suspension
Antigen- antibody conjugation
Fluorochrome & Fluorescence

9. Three main basis: Flow Cytometry: Basis Single cell suspension
Antigen- antibody conjugation
Fluorochrome & Fluorescence

10. Flow Cytometry: Basis
You need to have the cells flow one-by-one into the cytometer to do single cell analysis

11. Three main basis: Flow Cytometry: Basis Single cell suspension
Antigen- antibody conjugation
Fluorochrome &Fluorescent

12. Flow Cytometry: Basis Antigens Serve as cell identifiers
T-cells have surface antigens CD4 and CD8 on their surface
B-cells have surface antigens CD19, CD20, etc
Antigens
Cell surface antigens
B cell
T cell

13. Monoclonal Antibodies Polyclonal Antibodies
Flow Cytometry: Basis
Proteins used by the immune system to neutralize foreign invaders
Recognize, through specific binding antigens
Antibodies
Monoclonal Antibodies
Polyclonal Antibodies

14. Monoclonal antibodies Polyclonal antibodies
Flow Cytometry: Basis
Monoclonal antibodies
Immunochemically identical antibodies
React with a specific epitope on a given antigen
Polyclonal antibodies
Immunochemically dissimilar antibodies
React with various epitopes on a given antigen

15. Antigen- antibody conjugation Cell surface antigen receptors
Flow Cytometry: Basis
Antigen- antibody conjugation
Has many uses in the laboratory, including the specific identification of cells
Surface Staining: Direct Conjugation
Cell surface antigen receptors
Cell A B +
Incubation

16. Three main basis: Flow Cytometry: Basis Single cell suspension
Antigen- antibody conjugation
Fluorochrome & Fluorescence

17. Flow Cytometry: Basis Fluorochrome Fluorescent
Molecule gives out a particular color when specific laser light hits it
Fluorescent
Molecule loses energy and emits light

18. Flow Cytometry: Basis

19. Flow Cytometer: Components
Fluidics
Optical system
Electronics

20. Flow Cytometer: Components

21. Flow Cytometer: Components Flow Cytometry: Components

22. Flow cytometer: Light Scatter

23. Flow Cytometry: Process function
Detection of:
Phenotype
Cell cycle
Apoptosis
function

24. Flow cytometry: Measurable Parameters
Enzymatic activity
pH, intracellular ionized calcium, magnesium
Membrane fluidity
Apoptosis
Cell viability
Monitoring electropermeabilization of cells
Oxidative Burst
Characterizing multidrug resistance (MDR) in cancer cells
Glutathione
Various combinations (DNA/surface antigens etc.)

25. Flow Cytometer: Types BD FACSCalibur Partec Flow Cytometer BD FACSAria
BD FACSVantage
Moxi Flow™

26. BD FACSCanto II cytometer
Flow Cytometer: Types
BD FACSCanto II cytometer

27. 3. Acquisition process 4. Analysis of data Basics of flow cytometry
Main objectives: SAAI
By the end of this session you will learn:
Basics of flow cytometry
2. Staining process
3. Acquisition process
4. Analysis of data
5. Interpretation of data

28. Staining process

29. Staining Process: Main components
Take place in the processing Lab
Equipments
Antibodies
Reagents

30. Staining Types 1. Cell based staining Surface staining Intracellular
2. Cell free staining
Sample should be converted into single cell suspension prior to staining and acquisition

31. Staining Process: Surface staining protocol
Peripheral blood
sample collected in EDTA tube
Cells now are ready for acquisition

32. + Surface Staining: Direct Conjugation
Staining Process: Antigen- antibody conjugation
Surface Staining: Direct Conjugation
Cell surface antigen receptors
Cell A B +
Incubation

33. CD4 helper & CD8 Cytotoxic
Staining Process: Antigen- antibody conjugation
CD4 helper & CD8 Cytotoxic
CD: Cluster of Differentiation
The percentage of CD4 : CD8 = 2:1

34. 3. Acquisition process 4. Analysis of data Basics of flow cytometry
Main objectives: SAAI
By the end of this session you will learn:
Basics of flow cytometry
Staining process
3. Acquisition process
4. Analysis of data
5. Interpretation of data

35. Acquisition process

36. Acquisition Process Takes place in acquisition Lab Three main steps:
Pre-acquisition
Acquisition
Post acquisition

37. Acquisition Process Pre- acquisition:
Turn on the system
Open acquisition software
Run warming & priming of the system (Fluidics start up)
Make sure that priming process done successfully
System now is ready for acquisition

38. Acquisition Process 2. Acquisition: Create a file with your exp. name
Input your experiment layout
Load your samples in the carousel (autoloader)
Run the carousel and observe acquisition of the events on the worksheet

39. Acquisition Process
Use different gating strategies, to capture your interested population (monocytes, etc.) from total cell population
Select parameters to display (CD14 vs. CD33)
Once acquisition is complete, export your experiment data as FSC extensions

40. 3. Post acquisition: Run (Fluidics shut down) of the system
Acquisition Process
3. Post acquisition:
Run (Fluidics shut down) of the system
Now system is safe to shut down

41. Acquisition Process
Pre- acquisition:

42. Acquisition Process

43. Acquisition Process

44. Acquisition Process

45. Acquisition Process
2. Acquisition:

46. Acquisition Process

47. Acquisition Process

48. Acquisition Process

49. Acquisition Process

50. Acquisition Process

51. Acquisition Process

52. Acquisition Process

53. Acquisition Process

54. Acquisition Process

55. Acquisition Process

56. Acquisition Process

57. Acquisition Process

58. Acquisition Process

59. Acquisition Process

60. Acquisition Process

61. Acquisition Process

62. Acquisition Process

63. Acquisition Process

64. Acquisition Process

65. Acquisition Process
3. Post acquisition:

66. Acquisition Process

67. Acquisition Process

68. Questions

69. Acquisition process 4. Analysis of data Basics of flow cytometry
Main objectives: SAAI
By the end of this session you will learn:
Basics of flow cytometry
Staining process
Acquisition process
4. Analysis of data
5. Interpretation of data

70. Analysis
of data

71. Analysis Process: Main Steps
The computer programs can be used to analyze data subsequent to its acquisition
There are a variety of programs supplied for this purpose according to software acquisition
Whatever the program used, the principles of data analysis are the same

72. Analysis Process: FlowJo software
FlowJo is an open system for data analysis regardless acquisition software or data files extensions

73. Analysis Process: Light scatter
FSC vs. SSC
Forward scatter (FSC)
Side scatter
(SSC)
measures scattered light at 90 degrees to the laser path and measures the granularity of the cell
measures scattered light in the direction of the laser path and measures the size of the cell

74. Analysis Process : Gating strategy

75. Analysis process: Gating strategy
SSC-H
FSC-H

76. Analysis process: Gating strategy
Anti CD8
Anti CD4

77. Analysis Process: Gating strategy

78. Basics of flow cytometry Acquisition process Analysis of data
Main objectives: SAAI
By the end of this session you will learn:
Basics of flow cytometry
Staining process
Acquisition process
Analysis of data
Interpretation of data

79. Interpretation
of data

80. Value of flow cytometric data interpretation
Detection of
T-cell subsets
CD8 Cytotoxic T cell
CD4 Helper T cell

81. Data Interpretation
SSC-H
FSC-H

82. Data Interpretation
Anti CD8
Anti CD4

83. Questions

84. Practical Activities If you can, bring your lab coat Gp A 10.00 a.m
Gp B
10.30 a.m
Gp C
11.00 a.m
Gp D
11.30 a.m
Gp E
12.00 p.m
Gp F
12.30 p.m
If you can, bring your lab coat